Hello everybody, please, do you have some smart advice to solve this problem?
Here are two electrophoretograms from pulse field gel electrophoresis. In both experiments metagenomic DNA was being purified by washing out with many chemicals (including proteinase K, achromopeptidase, lysosym, cleaning buffers, formamid...) and further by PFGE. Later it was fragmented under the same conditions and with the same concentration of restriction endonucleases (Sau3AI, BamHI) in both experiments. At the beginning of each experiment the total amount of DNA was equal. DNA has been stored in agarose plugs for the whole period of purification and restriction fragmentation. I obtained these very different resultant electrophoretograms in these two experiements. And I do have this repeating problem already for some time! It seems that DNAse contaminated my sample and restricted the DNA to smaller fragments than I need. I am interested in restricted DNA pieces with the length ∼ 100 – 250 kb so I can cut it out from the gel for later use (picture exp.1). But instead I do get these different results often (picture exp.2).
1) Do you have experience what is the best way to remove DNAse contamination?
2) Do you think there can be some other problem?
Thank you for your help!
Tomas
Hi.....
What is the source of your metagenomic DNA preparation?
If it is soil then such pattern of restriction you may get due to DNAase activity and other contaminants that may damage the DNA. To check the possibility of existence of DNAase in your DNA preparation do simple test as
1.Digest your DNA with your choice of restriction enzyme
2. Take uncut DNA and incubate at tempt of restriction enzyme for the same duration
3. DNA with all components of restriction digestion except enzyme and incubate as same
After incubation load all 3 test sample on gel check the banding pattern.
If you get digestion / shearing like pattern in test no.2 then it is contaminated with DNAase
If you get digested pattern only in test 3 one of the component of restriction is contaminated.
else Another possibility is check the amount of enzyme and time length of incubation may be over digestion instead of doing complete digestion prefer partial digestion of DNA by either limiting enzyme or time or both.
Else check for PFGE gel running parameters.
Good luck.
Thank you for your answers, they were very useful. I found out that one of the reasons for bad digestion was my weak estimate for DNA concentration in gel. I now estimate DNA concentration in prestained edge of gel with computer software and apply diggestion according to the amount of DNA in not pre stained cut outs. It is more precise. Furthemore I limited possibility of DNAse contamination by very sterile conditions and enhanced the uniform spread of enzymes to DNA in gel by shaking added to restriction digestion.
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